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In vivo properties of lytic bacteriophages
isolates in Salmonella Enteritidis infected and
uninfected SPF chickens.
L. Fiorentin, N.D. Vieira and W. Barioni Jr.
Marcas da Embrapa em Inglês
Marcas do Ministério em Inglês
Brazilian Agricultural Research
Enterprise Embrapa Swine Poultry - BR 153, km
110, Vila Tamanduá, Caixa Postal 21
89700-000, Concórdia -SC, Brazil.
INTRODUCTION
RESULTS
Bacteriophages have recently been studied as a
new approach for reducing Salmonella infection
in chickens and therefore lowering the risks of
contamination of poultry products (Berchieri Jr
et al, 1991 Sklar et al., 2001). A perfect
understanding of phage biology within the poultry
alimentary tract, however, will be necessary for
a successful use of phages as therapeutic
agents, as well as to protect their longevity in
biocontrol. We have isolated and characterized
several bacteriophages lytic to Salmonella
Enteritidis and Salmonella Typhimurium (Fiorentin
et al., 2002). Three of these phages showing
different RAPD profiles were selected for
analyses of their invasiveness and rate of
excretion in SPF chickens.
Cloacal swabs of control birds (Group 3) were
negative for both bacteriophages and Salmonella
throughout the experiment. Swabs collected from
internal organs at necropsy of control birds were
also negative for both bacteriophages and
Salmonella. SE PT4 uninfected-phage administered
birds (Group 2) showed only 1/7 cloacal swabs
positive for bacteriophages one day after phage
administration and were negative throughout the
experiment. Swabs of SE PT4 infected-phage
administered chickens (group 1) were 7/7
bacteriophage positive two days after
administration and ranging from 4/7 to 7/7
thereafter (Figure 1). All birds in Group 1 were
positive at necropsy for both Salmonella and
bacteriophages in cecal contents while none was
bacteriophage positive for spleen and liver
samples, even though they were Salmonella
positive in all three samples. Cecal content
collected at necropsy showed 87.5 23.7 x 108
CFU/mL. All 35 isolates of Salmonella obtained
from cecal contents at necropsy of birds from
Group 1 remained sensitive to lysis by all three
phages.
MATERIAL AND METHODS
Bacteriophages were isolated from feces of free
range chickens and captive songbirds using a
field isolate of Salmonella Enteritidis PT4
(kindly provided by Dr. Paul Barrow, ARFC
Institute for Animal Health, Houghton Laboratory,
Cambridge, England) or Salmonella Typhimurium
ATCC 14028 as phage targets. Partial molecular
characterization of phage isolates has been
obtained and described elsewhere (Fiorentin et
al., 2002). The three phages denominated CNPSA1,
CNPSA3 and CNPSA4 selected for this study showed
icosahedrical head, dsDNA as nucleic acids and
had different RAPD profiles. Three groups of 7
SPF chickens (SPAFAS) were housed in isolator
cabinets under filtered air from birth till 16
days of age. Group 1 was orally infected at the
third day of age with 103 CFU of SE PT4 per bird
followed by oral inoculation two days later with
a pool of 105 PFU each phage per bird. Group 2
was only administered phage and Group 3 was kept
as uninfected control (Table 1). Cloacal swabs
were collected daily for SE PT4 and phage
isolation. Salmonella was isolated by incubating
swabs at 42ºC in peptonated Rappaport-Vasiliadis
broth overnight followed by iniculation in
Brilliant Green (BG) Agar at 37ºC and
identification of Salmonella by colony morphology
and serum agglutination with polivalent anti-O
reference serum. Bacteriophages were isolated by
incubating swabs at 37ºC in one milliliter of a
log phase SE PT4 culture, followed by a treatment
with 5 chloroform and inoculation of 5µL of
supernatant over a loan of SE PT4 grown in
nutrient broth solidified with 1 agarose. At
the 16th day of life all birds were necropsied
and attempts to isolate SE PT4 and
bacteriophages were made from cecal content,
spleen and liver of every bird. Five isolates of
Salmonella obtained from cecal content of each
bird in Group 1 were tested for their sensitivity
to the phages administered to the birds. Five
microliters of each phage was applied over a loan
of the Salmonella isolates grown in nutrient
broth solidified with 1 agarose. Cecal contents
were logarithmically diluted tenfold and
inoculated on BG with Novobiocin (40 µg/mL) to
obtain CFU/mL. Table 1. Experimental
design. Group Birds SE PT4 per bird1 Phage per
bird2 1 7 103 CFU at the 3rd day 105 PFU at
the 5th day 2 7 None 105 PFU at the 5th
day 3 7 None None 1 Field isolate. 2 PFU
of each of three phages.
CONCLUSIONS
We suggest that bacteriophages CNPSA1, CNPSA3 and
CNPSA4 do not remain in Salmonella-free birds
longer then one day while multiplying in
Salmonella-infected birds for longer periods.
Selection for phage resistant SE PT4 seemed not
to occur within the secretion period studied.
BIBLIOGRAPHY
Berchieri Jr., A., Lovell, M. A., Barrow, P.A.
The activity in the chicken alimentary tract of
bacteriophages lytic for Salmonella typhimurium.
Res. Microbiol., 142 541-549. 1991).
Fiorentin, L., Vieira, N.D. Barros, S.
Bacteriófagos líticos para Salmonella Enteritidis
e Salmonella Typhimurium isolados de fezes de
aves. In CONGRESSO BRASILEIRO DE MEDICINA
VETERINÁRIA, 29., 2002, Gramado, RS, Anais.
Gramado Sociedade Brasileira de Medicina
Veterinária, 2002. CD-ROM. Abstract PAN
1372. Sklar, B., Joerger, R.D. Attempts to
utilize bacteriophages to combat Salmonella
Enterica serovar Enteritidis infection in
chickens. J. Food Safety, 21 15-29. 2001.